Channels with cross-sectional thermal gradients

ABSTRACT

Provided herein are systems, devices, and methods for generating thermal gradients in channels and uses thereof. In particular, provided herein are system, methods, and devices employing first and second thermal layers positioned around a channel in order to create a thermal gradient across a cross-section of the channel having, for example, a nucleic acid denaturation zone, a nucleic acid annealing zone, and a nucleic acid polymerization zone. Such devices find use in, for example, nucleic acid amplification procedures, including digital polymerase chain reaction (dPCR) to temperature cycle droplets for amplification of nucleic acid templates within the droplets.

The present application is a divisional of U.S. application Ser. No.13/724,503, filed Dec. 21, 2012, which claims priority to U.S.provisional application Ser. No. 61/582,035, filed Dec. 30, 2011, theentirety of each of which is herein incorporated by reference.

FIELD OF THE INVENTION

Provided herein are systems, devices, and methods for generating thermalgradients in channels and uses thereof. In particular, provided hereinare systems, methods, and devices employing first and second thermallayers positioned around a channel in order to create a thermal gradientacross a cross-section of the channel having, for example, a nucleicacid denaturation zone, a nucleic acid annealing zone, and a nucleicacid polymerization zone. Such devices find use in, for example, nucleicacid amplification procedures, including digital polymerase chainreaction (dPCR) to temperature cycle droplets for amplification ofnucleic acid templates within the droplets.

BACKGROUND

The temperature dependence of biochemical and chemical reaction ratesposes a particular challenge to efforts to improve reaction efficiencyand speed by miniaturization. A time-domain approach, whereby not onlythe reaction volume but also the entire housing is kept at a desiredtemperature, is only suitable for isothermal conditions. If temperatureneeds to be changed or cycled in a rapid and controlled manner, theadded thermal mass of the housing limits the rate and/or precision thatcan be achieved.

In the space-domain approach (see, e.g., Kopp, et al., Science 1998,280, 1046-1048; Burns, et al., Science 1998, 282, 484-487; Chiou et al.,Anal. Chem. 2001, 73, 2018-2021; and Nakano et al., Biosci. Biotechnol.Biochem. 1994, 58, 349-352), different parts of the reaction housing arekept at different temperatures, and reaction volume is brought inthermal contact with a desired part of the housing to keep it at thetemperature of that part. If necessary, the reaction volume can then bemoved to a different part of the housing to change the temperature; and,depending on the trajectory of the reaction volume, the temperatureprofile of it can be adjusted or cycled as desired. In the art, most ofthe implementations of the space-domain dynamic thermal control havebeen directed to miniaturized PCR thermocycling. Continuous meanderingor spiral channels laid across temperature zones have been demonstratedfor continuous flow through amplification (see, e.g., Fukuba et al.,CHEMICAL ENGINEERING JOURNAL 101 (1-3): 151-156 Aug. 1, 2004);direct-path arrangements with a reaction slug moving back and forth havebeen described (see, e.g., Chiou et al., Anal. Chem. 2001, 73,2018-2021); and finally, cycling of an individual reaction through aloop has been demonstrated (see, e.g., Jian Liu Markus Enzelberger etal. Electrophoresis 2002, 23, 1531-1536).

SUMMARY OF THE INVENTION

Provided herein are systems, devices, and methods for generating thermalgradients in channels (e.g., microchannels) and uses thereof. Inparticular, provided herein are system, methods, and devices employingfirst and second thermal layers positioned around a channel in order tocreate a thermal gradient across a cross-section of the channel having,for example, a nucleic acid denaturation zone, a nucleic acid annealingzone, and a nucleic acid polymerization zone. Such devices find use in,for example, nucleic acid amplification procedures, including digitalpolymerase chain reaction (dPCR) to temperature cycle droplets foramplification of nucleic acid templates within the droplets or otherpartitions.

In some embodiments, provided herein are devices and systems comprising:a) a channel (e.g., microchannel) comprising a surface configured fortransmission of a carrier medium comprising droplets or partitions, b) afirst thermal layer positioned along the microchannel configured to heata portion of the channel surface to a nucleic acid denaturationtemperature; and c) a second thermal layer positioned along the channelconfigured to heat or cool a portion of the channel surface to a nucleicacid annealing temperature; wherein the channel and first and secondthermal layers are positioned to create a thermal gradient across across-section of the channel, and wherein the thermal gradient comprisesa nucleic acid denaturation zone, a nucleic acid annealing zone, and anucleic acid polymerization zone.

In certain embodiments, the devices and systems further comprise aplurality of motion components positioned along the channel (e.g.,microchannel), wherein the plurality of motion components are configuredto impart motion to the droplets or partitions in the carrier mediumwhen present in the channel such that, for example, random stochasticcontact of the droplet or partition with the channel surface isincreased. In particular embodiments, motion is imparted via mechanicalagitation. In some embodiments, the mechanical agitation is selectedfrom the group consisting of: vibration, low-frequency sonication, andacoustic waves. In some embodiments, the mechanical agitation isimparted with solenoid valves or mechanical switches. In furtherembodiments, the motion is imparted via convection and/or turbulentflow. In additional embodiments, the motion is imparted via anelectrical field. In particular embodiments, the devices and systemsfurther comprise the carrier medium comprising the droplets orpartitions. In additional embodiments, the plurality of motioncomponents are positioned intermittently along the channel. In otherembodiments, the plurality of motion components are positionedcontinuously along the channel.

In some embodiments, provided herein are devices and systems comprising:a) a channel (e.g., microchannel) comprising a surface configured fortransmission of a carrier medium comprising droplets or partitions, b) afirst thermal layer positioned along the microchannel configured to heata portion of the channel surface to a nucleic acid denaturationtemperature; c) a second thermal layer positioned along the channelconfigured to heat or cool a portion of the channel surface to a nucleicacid annealing temperature; and d) a plurality of motion componentspositioned along the channel (e.g., microchannel), wherein the pluralityof motion components are configured to impart motion (e.g., motion isimparted via mechanical agitation, electrical field, and/or viaconvection and/or turbulent flow) to the droplets or partitions in thecarrier medium when present in the channel such that, for example,random stochastic contact of the droplet or partition with the channelsurface is increased (e.g., where the plurality of motion components arepositioned intermittently or continuously along the channel); andwherein the channel and first and second thermal layers are positionedto create a thermal gradient across a cross-section of the channel, andwherein the thermal gradient comprises a nucleic acid denaturation zone,a nucleic acid annealing zone, and a nucleic acid polymerization zone.

In particular embodiments, provided herein are devices and systemscomprising: a) a channel (e.g., microchannel) comprising a surfaceconfigured for transmission of a liquid or gas carrier medium (e.g.,mineral oil or nitrogen gas) comprising droplets or partitions (e.g.,where the droplets or partitions comprise primers, such as amplificationprimers; and/or comprise dNTPs; and/or comprise a polymerase), b) afirst thermal layer positioned along the microchannel configured to heata portion of the channel surface to a nucleic acid denaturationtemperature; and c) a second thermal layer positioned along the channelconfigured to heat or cool a portion of the channel surface to a nucleicacid annealing temperature; wherein the channel and first and secondthermal layers are positioned to create a thermal gradient across across-section of the channel, and wherein the thermal gradient comprisesa nucleic acid denaturation zone, a nucleic acid annealing zone, and anucleic acid polymerization zone.

In further embodiments, provided herein are devices and systemscomprising: a) a channel (e.g., microchannel) comprising a surfaceconfigured for transmission of a carrier medium comprising droplets orpartitions, b) a first thermal layer positioned along the microchannelconfigured to heat a portion of the channel surface to a nucleic aciddenaturation temperature; c) a second thermal layer positioned along thechannel configured to heat or cool a portion of the channel surface to anucleic acid annealing temperature; and d) optical array sensorpositioned along the channel configured to monitor signal output in thedroplets or partitions (e.g., the optical array sensor comprises acomponent selected from the group consisting of: a light-emitting diode,a photodiode array, an optical fiber bundle, a laser configured forfluorometry, a piezoelectric crystal, an interdigital cantilever, aphotovoltaic cell, and a gated linear CCD array), wherein the channeland first and second thermal layers are positioned to create a thermalgradient across a cross-section of the channel, and wherein the thermalgradient comprises a nucleic acid denaturation zone, a nucleic acidannealing zone, and a nucleic acid polymerization zone.

In particular embodiments, the carrier medium comprises liquid (e.g.,mineral oil). In further embodiments, the carrier medium comprises gas(e.g., nitrogen gas). In particular embodiments, the droplets orpartitions each comprise primers (e.g., amplification primers) and anucleic acid template. In other embodiments, the droplets or partitionsfurther comprise a polymerase and dNTPs. In some embodiments, thesystems and devices further comprise an optical array sensor positionedalong the channel configured to monitor signal output in the droplets orpartitions. In certain embodiments, the optical array sensor comprises acomponent selected from the group consisting of: a light-emitting diode,a photodiode array, an optical fiber bundle, a laser configured forfluorometry, a piezoelectric crystal, an interdigital cantilever, aphotovoltaic cell, and a gated linear CCD array.

In certain embodiments, provided herein are devices and systemscomprising: a) a channel (e.g., microchannel) comprising a surfaceconfigured for transmission of a carrier medium comprising droplets orpartitions, b) a first thermal layer positioned along the microchannelconfigured to heat a portion of the channel surface to a nucleic aciddenaturation temperature (e.g., about 90-110 degrees Celsius, such asabout 90 . . . 95 . . . 100 . . . 100 . . . 105 . . . or about 110degrees, or higher); and c) a second thermal layer positioned along thechannel configured to heat or cool a portion of the channel surface to anucleic acid annealing temperature (e.g., about 20-60 degrees Celsius,such as about 20. . . 30 . . . 40 . . . 50 . . . or 60 degrees Celsius);wherein the channel and first and second thermal layers are positionedto create a thermal gradient across a cross-section of the channel, andwherein the thermal gradient comprises a nucleic acid denaturation zone,a nucleic acid annealing zone, and a nucleic acid polymerization zone(e.g., which has a temperature of about 60-90 degrees Celsius, such asabout 60 . . . 70 . . . 80 . . . or 90 degrees Celsius).

In some embodiments, the denaturation temperature is about 90-110degrees Celsius (e.g., about 90 . . . 95 . . . 100 . . . 100 . . . 105 .. . or about 110 degrees, or higher). In particular embodiments, theannealing temperature is about 20-60 degrees Celsius (e.g., about 20 . .. 30 . . . 40 . . . 50 . . . or 60 degrees Celsius). In particularembodiments, the nucleic acid polymerization zone has a temperature ofabout 60-90 degrees Celsius (e.g., 60 . . . 70 . . . 80 . . . or 90degrees Celsius).

In some embodiments, provided herein are devices and systems comprising:a) a channel (e.g., microchannel) comprising a surface configured fortransmission of a carrier medium comprising droplets or partitions(e.g., where the droplets or partitions comprise an emulsion or amacromolecular assembly selected from the group consisting of:unilamellar vesicles, bilamellar vesicles, micelles, liposomes,nanostructures, macromolecular cages, carbon nanotubes, fullerenes orparamagnetic particles), b) a first thermal layer positioned along themicrochannel configured to heat a portion of the channel surface to anucleic acid denaturation temperature; and c) a second thermal layerpositioned along the channel configured to heat or cool a portion of thechannel surface to a nucleic acid annealing temperature; wherein thechannel and first and second thermal layers are positioned to create athermal gradient across a cross-section of the channel, and wherein thethermal gradient comprises a nucleic acid denaturation zone, a nucleicacid annealing zone, and a nucleic acid polymerization zone.

In some embodiments, the droplets or partitions comprise an emulsion ora macromolecular assembly selected from the group consisting of:unilamellar vesicles, bilamellar vesicles, micelles, liposomes,nanostructures, macromolecular cages, carbon nanotubes, and fullerenes.

In certain embodiments, the droplets or partitions comprise paramagneticmicroparticles. In further embodiments, the motion component induces anoscillating magnetic field along the channel.

In certain embodiments, provided herein are methods comprising: a)providing a system or device comprising: i) a channel (e.g.,microchannel) comprising a surface configured for transmission of acarrier medium comprising droplets or partitions, ii) a first thermallayer positioned along the channel configured to heat a portion of thechannel surface to a nucleic acid denaturation temperature; and iii) asecond thermal layer positioned along the channel configured to heat orcool a portion of the channel surface to a nucleic acid annealingtemperature; wherein the channel and first and second thermal layers arepositioned to create a thermal gradient across a cross-section of thechannel, and wherein the thermal gradient comprises a nucleic aciddenaturation zone, a nucleic acid annealing zone, and a nucleic acidpolymerization zone; and b) flowing the carrier medium through thechannel such that the droplets or partitions repeatedly pass through thethermal gradient causing temperature cycling in the droplets orpartitions, wherein the droplets or partitions each comprise primers, anucleic acid template, a polymerase, nucleotides, salts, and buffer, andwherein the temperature cycling causes amplification of the nucleic acidtemplates in the droplets or partitions.

In some embodiments, provided herein are methods comprising: a)providing a system or device comprising: i) a channel (e.g.,microchannel) comprising a surface configured for transmission of acarrier medium comprising droplets or partitions, ii) a first thermallayer positioned along the channel configured to heat a portion of thechannel surface to a nucleic acid denaturation temperature; iii) asecond thermal layer positioned along the channel configured to heat orcool a portion of the channel surface to a nucleic acid annealingtemperature; and iv) a plurality of motion components positioned alongthe channel (e.g., microchannel), wherein the plurality of motioncomponents are configured to cause the droplets to repeatedly passthrough the thermal gradient by imparting motion to the droplets (e.g.,motion is imparted via mechanical agitation, electrical agitation, orconvection and/or turbulent flow); wherein the channel and first andsecond thermal layers are positioned to create a thermal gradient acrossa cross-section of the channel, and wherein the thermal gradientcomprises a nucleic acid denaturation zone, a nucleic acid annealingzone, and a nucleic acid polymerization zone; and b) flowing the carriermedium through the channel such that the droplets or partitionsrepeatedly pass through the thermal gradient causing temperature cyclingin the droplets or partitions, wherein the droplets or partitions eachcomprise primers, a nucleic acid template, a polymerase, nucleotides,salts, and buffer, and wherein the temperature cycling causesamplification of the nucleic acid templates in the droplets orpartitions.

In certain embodiments, the device used in the methods further comprisesa plurality of motion components positioned along the channel (e.g.,microchannel), wherein the plurality of motion components are configuredto cause the droplets to repeatedly pass through the thermal gradient byimparting motion to the droplets. In other embodiments, the motion isimparted via mechanical agitation. In other embodiments, the mechanicalagitation is selected from the group consisting of: vibration,low-frequency sonication, and acoustic waves. In certain embodiments,the mechanical agitation is imparted with solenoid valves or mechanicalswitches. In other embodiments, the motion is imparted via convectionand/or turbulent flow. In further embodiments, the motion is impartedvia an electrical field. In some embodiments, the plurality of motioncomponents are positioned intermittently along the channel.

In certain embodiments, provided herein are methods comprising: a)providing a system or device comprising: i) a channel (e.g.,microchannel) comprising a surface configured for transmission of acarrier medium comprising droplets or partitions, ii) a first thermallayer positioned along the channel configured to heat a portion of thechannel surface to a nucleic acid denaturation temperature; iii) asecond thermal layer positioned along the channel configured to heat orcool a portion of the channel surface to a nucleic acid annealingtemperature; and iv) an optical array sensor positioned along thechannel (e.g., microchannel); wherein the channel and first and secondthermal layers are positioned to create a thermal gradient across across-section of the channel, and wherein the thermal gradient comprisesa nucleic acid denaturation zone, a nucleic acid annealing zone, and anucleic acid polymerization zone; b) flowing the carrier medium throughthe channel such that the droplets or partitions repeatedly pass throughthe thermal gradient causing temperature cycling in the droplets orpartitions, wherein the droplets or partitions each comprise primers, anucleic acid template, a polymerase, nucleotides, salts, and buffer, andwherein the temperature cycling causes amplification of the nucleic acidtemplates in the droplets or partitions; and c) monitoring the templateamplification with the optical array sensor.

In particular embodiments, the device further comprises an optical arraysensor positioned along the channel (e.g., microchannel), and whereinthe method further comprises monitoring the template amplification withthe optical array sensor. In other embodiments, wherein the opticalarray sensor comprises a component selected from the group consistingof: a light-emitting diode, a photodiode array, an optical fiber bundle,a laser configured for fluorometry, a piezoelectric crystal, aninterdigital cantilever, a photovoltaic cell, and a gated linear CCDarray.

In certain embodiments, provided herein are methods comprising:performing digital PCR amplification with droplets, wherein the dropletcomprises at least one macromolecular assembly selected from the groupconsisting of: unilamellar vesicles, bilamellar vesicles,micelles,liposomes, nanostructures, macromolecular cages, carbon nanotubes, andfullerenes. In some embodiments, the methods further comprise sequencingthe amplified templates in at least some of the droplets. In someembodiments, flowing the carrier medium through the channel is performedin a continuous manner, a pulsed manner, a batch manner, or fed-batchmanner.

In some embodiments, provided herein are methods of analyzing nucleicacid comprising: a) separating a nucleic acid sample into a plurality ofpartitions, wherein the nucleic acid sample comprises a mixture ofnucleic acid molecules and amplification reagents, wherein a portion ofthe plurality of partitions are single nucleic acid molecule containingpartitions, and a portion of the plurality of partitions are zeronucleic acid molecule containing partitions, and the number ofpartitions containing more than one nucleic acid molecule is zero or astatistically insignificant fraction of the total number of partitions,and wherein the portions comprise at least one macromolecular assemblyselected from the group consisting of: unilamellar vesicles, bilamellarvesicles,micelles, liposomes, nanostructures, macromolecular cages,carbon nanotubes, and fullerenes; b) treating the plurality ofpartitions under amplification conditions such that the single nucleicacid molecule containing partitions become amplicon-containingpartitions; and c) physically sorting the plurality of partitions.

DESCRIPTION OF THE FIGURES

FIG. 1 shows a side view of an exemplary microchannel, with a firstthermal layer on ‘top’ of the microchannel (configured to heat to 95degrees Celsius) and a second thermal layer on the ‘bottom’ of themicrochannel (configured to heat or cool to 50 degrees Celsius),creating a temperature gradient with a middle zone at 72 degreesCelsius.

FIG. 2 shows a side view of a microchannel with various macromolecularstructures that could be used, for example, with digital PCRamplification methods.

FIG. 3 shows a side view of an exemplary microfluidic channel withvarious components to create turbulent flow, including barriers, ridges,switches, and valves.

FIG. 4 shows an exemplary optical array sensor along a microchannel.

DEFINITIONS

As used herein, the term “partition” refers to a volume of fluid (e.g.liquid or gas) that is a separated portion of a bulk volume. A bulkvolume may be partitioned into any suitable number (e.g. 10 ² . . . 10³. . . 10⁴ . . . 10⁵ . . . 10⁶ . . . 10⁷, etc.) of smaller volumes (i.e.partitions). Partitions may be separated by a physical barrier or byphysical forces (e.g. surface tension, hydrophobic repulsion, etc.).Partitions generated from the larger volume may be substantially uniformin size (monodisperese) or may have non-uniform sizes (polydisperse).Partitions may be produced by any suitable manner (e.g. emulsion,microfluidics, microspray, etc.). Exemplary partitions are droplets.

As used herein, the term “droplet” refers to a small volume of liquidthat is immiscible with its surroundings (e.g. gases, liquids, surfaces,etc.). A droplet may reside upon a surface, be encapsulated by a fluidwith which it is immiscible (e.g. the continuous phase of an emulsion, agas (e.g. air, nitrogen)), or a combination thereof. A droplet istypically spherical or substantially spherical in shape, but may benon-spherical. The shape of an otherwise spherical or substantiallyspherical droplet may be altered by deposition onto a surface. A dropletmay be a “simple droplet” or a “compound droplet,” wherein one dropletencapsulates one or more additional smaller droplets. The volume of adroplet and/or the average volume of a set of droplets provided hereinis typically less than about one microliter (e.g. 1 μL . . . 0.1 μL . .. 10 nL . . . 1 nL . . . 100 pL . . . 10 pL . . . 1 pL . . . 100 fL . .. 10 fL . . . 1 fL). The diameter of a droplet and/or the averagediameter of a set of droplets provided herein is typically less thanabout one millimeter (e.g. 1 mm . . . 100 μm . . . 10 μm . . . 1 μm).Droplets may be formed by any suitable technique (e.g. emulsification,microfluidics, etc.) and may be monodisperse (e.g., substantiallymonodisperse) or polydisperse.

As used herein, the term “packet” refers to a set of droplets or otherisolated partitions disposed in the same continuous volume, in the sameregion of a continuous volume, on the same surface, or otherwisegrouped. A packet may constitute all of the droplets of bulk volume(e.g. an emulsion), or a segregated fraction of droplets from a bulkvolume (e.g. at a range of positions along a channel, containing thesame target amplicon, etc.). A packet may constitute all the dropletslocated along a surface (e.g. chip or microfluidic surface), or thedroplets in a defined region of a surface. A packet may refer to a setof droplets that when analyzed in partial or total give a statisticallyrelevant sampling for quantitative analysis of the entire startingsample (e.g. the entire bulk volume).

As used herein, the term “amplifying” or “amplification” in the contextof nucleic acids refers to the production of multiple copies of apolynucleotide, or a portion of the polynucleotide, typically startingfrom a small amount of the polynucleotide (e.g., a single polynucleotidemolecule), where the amplification products or amplicons are generallydetectable or can be made detectable. Amplification of polynucleotidesencompasses a variety of chemical and enzymatic processes. Thegeneration of multiple DNA copies from one or a few copies of a targetor template DNA molecule during a polymerase chain reaction (PCR) or aligase chain reaction (LCR) are forms of amplification. Amplification isnot limited to the strict duplication of the starting molecule. Forexample, the generation of multiple cDNA molecules from a limited amountof RNA in a sample using reverse transcription (RT)-PCR is a form ofamplification. Furthermore, the generation of multiple RNA moleculesfrom a single DNA molecule during the process of transcription is also aform of amplification.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, that is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product that is complementary to a nucleic acid strand isinduced (e.g., in the presence of nucleotides and an inducing agent suchas a biocatalyst (e.g., a DNA polymerase or the like) and at a suitabletemperature and pH). The primer is typically single stranded for maximumefficiency in amplification, but may alternatively be double stranded.If double stranded, the primer is generally first treated to separateits strands before being used to prepare extension products. In someembodiments, the primer is an oligodeoxyribonucleotide. The primer issufficiently long to prime the synthesis of extension products in thepresence of the inducing agent. The exact lengths of the primers willdepend on many factors, including temperature, source of primer and theuse of the method.

As used herein, the term “sample” refers to anything capable of beinganalyzed by the methods provided herein. In some embodiments, the samplecomprises or is suspected to comprise one or more nucleic acids capableof analysis by the methods. Preferably, the samples comprise nucleicacids (e.g., DNA, RNA, cDNAs, etc.). Samples may be complex samples ormixed samples, which contain nucleic acids comprising multiple differentnucleic acid sequences. Samples may comprise nucleic acids from morethan one source (e.g. difference species, different subspecies, etc.),subject, and/or individual. In some embodiments, the methods providedherein comprise purifying the sample or purifying the nucleic acid(s)from the sample. In some embodiments, the sample contains purifiednucleic acid. In some embodiments, a sample is derived from abiological, clinical, environmental, research, forensic, or othersource.

DETAILED DESCRIPTION

Provided herein are systems, devices, and methods for generating thermalgradients in channels (e.g., microchannels) and uses thereof. Inparticular, provided herein are system, methods, and devices employingfirst and second thermal layers positioned around a channel in order tocreate a thermal gradient across a cross-section of the channel having,for example, a nucleic acid denaturation zone, a nucleic acid annealingzone, and a nucleic acid polymerization zone. Such devices find use in,for example, nucleic acid amplification procedures, including digitalpolymerase chain reaction (dPCR) to temperature cycle droplets foramplification of nucleic acid templates within the droplets or otherpartitions.

In some embodiments, a sample is analyzed for the presence and/orabundance of target nucleic acid sequences. In some embodiments, thetarget nucleic acid is present in a potentially complex sample that maycontain many different nucleic acid sequences, each of which may or maynot contain the target sequence. In some embodiments, a sample isanalyzed to determine the proportion of nucleic acid moleculescontaining a target sequence of interest. In some embodiments, a complexsample is analyzed to detect the presence and/or measure the abundanceor relative abundance of multiple target sequences. In some embodiments,methods provided herein are used to determine what sequences are presentin a mixed sample and/or in what relative proportions.

Provided herein are channels (e.g., microchannels) with thermalgradients that allow for amplification of templates in droplets.Generally, continuous-flow microfluidic PCR systems require a liquid orgaseous carrier (such as mineral oil or inert gas) to provide bulk fluidor aerosol transport of discrete aqueous phase reaction elements. Incertain embodiments, the methods described herein employ oil dispersionor aerosolization to create discrete aqueous phase nanodroplets fortransport through microfluidic channels imbedded in a fluid cell device.Droplets containing both DNA targets (amplicons) and amplificationreagents (polymerase, dNTPs, buffer, and salt) can be prepared directlyin either a biphasic liquid or gaseous carrier stream that is readilydispensed into a microfluidic chamber for subsequent amplification.

In certain embodiments, the methods and devices described herein utilizemechanical agitation to provide intermittent mixing pulses along amicrofluidic channel, such that random stochastic contact with surfacewalls provides sufficient thermal cycling for DNA amplification ofsequestered suspensions (e.g., bi-phasic nanodroplets, aerosols, and/ormacromolecular assemblies). By controlling microchannel architecture(e.g., path length), virtually any desired sensitivity (e.g., copies permL) may be attained. In other embodiments, the approach described hereinutilizes convection and/or turbulent flow to provide mixing, such thatrandom stochastic contact with surface walls provides sufficient thermalcycling for DNA amplification of sequestered suspensions (e.g.,bi-phasic nanodroplets, aerosols, and/or macromolecular assemblies).

In certain embodiments, the methods and devices described herein employa continuous optical array sensor for monitoring of microfluidic PCRapplications. Microfluidic PCR systems (e.g., end-point or real-time)can employ a variety of detection strategies. One approach describedherein employs a continuous optical array sensor to monitor signaloutput along the length of a microfluidic channel.

In certain embodiments, provided herein are methods and devices forrapid, bi-thermal PCR amplification enclosed in a miniaturized fluidicdevice, providing output for downstream molecular analysis (e.g.,flow-cytometry, mass spectrometry, and or microcapillaryelectrophoresis). In certain embodiments, provided herein are methodsfor PCR amplification utilizing two distinct isothermal layers (e.g.,denaturation zone and annealing zone) to create a temperature gradientacross specialized microfluidic channels designed to cycle small-volumefluid elements through the thermal temperature extremes of DNAdenaturation (e.g., ˜95° C.) and annealing (e.g., ˜50° C.). Flow throughthe bi-thermal microfluidic device may be, for example, continuous orpulsed in order to best optimize/synchronize controlled fluid movementand requisite heat transfer. Bulk fluid displacement may be accomplishedusing a carrier comprised of liquid (mineral oil) or gas (nitrogen),driven by mechanical pressure (or vacuum). The precise geometricarchitecture of the microfluidic cell is configured to accommodateamplification of small-size (e.g., 100-500 bp) amplicons that requireshort primer-extension/elongation times (e.g., polymerization rate of˜300 nucleotides/second) and may be configured to accommodate largeramplicon targets (e.g., 2 Kb or longer).

In certain embodiments, amplification proceeds by conventional means asdiscrete fluid elements cycle along channels embedded between theopposing isothermal layers, obviating the need for active/dynamictemperature cycling and control. The microfluidic channels are designedto transport the discrete fluid elements through the differentialthermal zones in a temporal and spatial manner that sustains thetraditional amplification cycle (denaturation, annealing, and extension)and provides uniform and efficient heat transfer for rapid cycling;thereby significantly reducing the overall target amplification time andminimizing the physical system dimensions (footprint). The fluid cell isdesigned to carry the samples through the three amplification zones in arapid manner that provides adequate residence times for each of theseparate temperature-dependent steps (denaturation, annealing, andcatalysis). Sample throughput is limited only by the number ofmicrocapillary channels that are fabricated and interrogated. In certainembodiments, the detection scheme may be, but is not limited to:fluorescence based end-point PCR analysis (e.g., flow cytometry ormicrocapillary-based detection strategies), label-free end-point PCR(e.g., mass spectrometry), and fluorescence-based Real Time PCR analysis(e.g., utilizing on-board optical detection strategies). Flowcytometry-based detection strategies are best suited forFluorescence-based end-point PCR analysis, by leveraging the inherentstratification afforded by fluid cell transport. Alternate detectionstrategies such as microcapillary electrophoresis or conductance-basedmethods may also be employed. Mass spectrometry is ideally suited forlabel-free end-point PCR analysis.

In other embodiments, real time PCR applications are employed. Real timedetection generally employs a network of optical fluorescencedetectors/sensors arrayed around the microcapillary channels in a mannerthat captures the spatially-distributed, time-resolved fluorescenceprofile of a discrete amplification reaction.

In certain embodiments, carrier flow through the microfluidic device maybe continuous, pulsed, batch, or fed-batch in order to bestoptimize/synchronize controlled fluid movement and requisite heattransfer for thermal cycling. In order to minimize sample cross-talkbetween discrete volume entities (containing reactants and separate,discrete amplicons/targets) transported through the amplificationchamber, many factors may be controlled including (but not limited to):the size-distribution of the nanodroplets, the concentration of thedispersed nanodroplets in the carrier medium, the flow-rate, andpotentially the use of cleaning agents that can be introduced in betweenserial sample addition steps. Many advantages are conferred by thisapproach such as: minimizing reagent consumption and costs by reducingamplification reaction volumes to nanoliter or picoliter ranges,expediting heat transfer for rapid thermal cycling, reducing the energyburden for temperature control and heat transfer, increasing samplethroughput, and miniaturizing the overall system footprint. Togetherwith the bi-isothermal PCR amplification/detection process describedabove, this approach provides a strategy for a next-generation,continuous-flow, microfluidic amplification system for end-point andreal-time PCR applications, among others.

In certain embodiments, the approach described herein utilizesmechanical agitation (e.g., vibration, low-frequency mild sonication,acoustic waves, solenoid valves, nanomechanical switches, etc.) toprovide intermittent mixing pulses along a microfluidic channel, suchthat random stochastic contact with temperature gradient (e.g., ˜50° C.-95° C.) extremes located along the microfluidic channel surface wallprovides sufficient thermal cycling for DNA amplification of sequesteredsuspensions (e.g., bi-phasic nanodroplets, aerosols, and/ormacromolecular assemblies encapsulating discrete PCR reactions).Physical channel-length can be manipulated to achieve the desired levelof amplification. Since overall amplicon target copy-number in clinicalsamples are expected to vary over a wide dynamic range, channel lengthscan be set to detect different concentrations of target (e.g., as low as100 targets per mL of blood) by imbedding microfluidic paths of varyinglength. Carrier flow through the microfluidic PCR device may be, forexample, continuous, pulsed, batch, or fed-batch in order to bestoptimize/synchronize controlled fluid movement and requisite heattransfer for thermal cycling. Many advantages are conferred by thisapproach, including: minimizing reagent consumption and costs byreducing amplification reaction volumes (e.g., microliter to picoliterrange) increasing both assay sensitivity and specificity by signalconcentration (e.g., afforded by volume sequestration), expediting heattransfer for rapid thermal cycling, reducing the energy burden fortemperature control and heat transfer, increasing sample throughput, andminiaturizing the overall system footprint.

In some embodiments, microencapsulated macromolecular assemblies(including, but not limited to: unilamellar vesicles, bilamellarvesicles, micelles, liposomes, nanostructures, macromolecular cages,carbon nanotubes, and fullerenes) are used as self-contained reactionvessels for use in end-point and/or real time microfluidic PCRapplications. Such embodiments employ discrete reaction vessels in acarrier suspension stream. Each microencapsulated vessel contains anamplicons/target in combination with the PCR master-mix required fortemplate amplification. Sequestration of a single polymerase andamplicon DNA target in the presence of primers, deoxynucleotidetriphosphates, and fluor-conjugate label should result in both increasedassay sensitivity (e.g., due to the volume-localization of the amplifiedsignal) and increased specificity (e.g., as small-volume localizationinherently reduces the starting template copy-number). Many advantagesare conferred by this approach such as: increased sensitivity, increasedspecificity, minimizing reagent consumption and costs by reducingamplification reaction volumes to nanoliter or picoliter ranges,expediting heat transfer for rapid thermal cycling, reducing the energyburden for temperature control and heat transfer, increasing samplethroughput, and miniaturizing the overall system footprint.

In certain embodiments, by incorporating ridges, barriers, or switches,both convection and turbulent flow can be enhanced to deliver adequatethermal contact with the microfluid channel walls.

In some embodiments, the systems, devices, and methods employ acontinuous optical array sensor to monitor PCR signal output along thesurface of a microfluidic channel. Optical detection can be accomplishedin any number of manners, including, for example: LED (light-emittingdiode) photodiode arrays, optical fiber bundles, laser inducedfluorometry, piezoelectric crystals, interdigital cantilevers,photovoltaic cells, and gated linear CCD arrays.

The present invention is not limited by the types of samples that areassayed according to the methods disclosed herein and can include, forexample, serum, plasma, lymphatic fluid, interstitial fluid,cerebrospinal fluid (CSF), amniotic fluid, seminal fluid, vaginalexcretions, serous fluid, synovial fluid, pericardial fluid, peritonealfluid, pleural fluid, transudates, exudates, cystic fluid, bile, gastricfluids, intestinal fluids, saliva, urine, breast milk, and teardrops.

In certain embodiments, the droplets may comprise paramagneticmicroparticles (PMPs). In such embodiments, one can employ anoscillating magnetic field to attract droplets containing PMPs to thedesired temperature surface. In certain embodiments, the oscillatingmagnetic field frequency is tuned so that PMP-containing droplets cantraverse the thermal regions in a cyclical manner that promotes rapidand efficient PCR amplification. In certain embodiments, the thermalzones are magnetic. However, it is not necessary that the thermal zones,themselves, be magnetic. Rather, in some embodiments, it is practical toapply an external magnetic field to the surroundings. In such aconfiguration, the PMP-droplets transversely flow through the system(continuous-flow) as they are repeatedly attracted to the desiredtemperature surface (magnetically driven PCR cycling). In otherembodiments, a static “batch” approach is utilized that does not utilizecontinuous-flow. In some embodiments, it is possible to pause themagnetic field briefly, before oscillation/inversion, so that thePMP-droplets can diffuse towards the intermediate temperature zone forannealing. In general, it follows that the strength and magnitude of themagnetic field is directly proportional to the PMP-droplet migrationrate; and that the duration of the magnetic field phase is proportionalto thermal “hold” step times. Another advantage of PMP-droplets is thatthey may be easily manipulated and collected downstream using magneticfields.

In some embodiments, one can use a macromolecular cage to partition andconfine reaction elements. Macromolecular cages may also be derivatizedwith paramagnetic materials that impart a response in a magnetic field.

In certain embodiments, droplet migration is driven electronically byusing an electrical field to draw the drops towards a thermal layer. Infurther embodiments, macromolecular cages are derivatized with chargedfunctional groups to promote desired migration in the electric field.

In some embodiments, all or a portion of the nucleic acid to be analyzedor manipulated is partitioned, for example, from different nucleic acidmolecules or from other copies of the same nucleic acid molecule.Nucleic acid molecules may be segregated into droplets and/or may beassociated with solid surfaces (e.g., beads) that are themselvessegregated from one another. In some embodiments, provided herein isamplification (e.g. PCR amplification) of the partitioned nucleic acidsof a sample. In some embodiments, amplification reagents (e.g. primers)are added to a sample prior to partitioning and/or concurrent withpartitioning, or amplification reagents are added to the partitionedsample. In some embodiments, primers are hybridized to template nucleicacids prior to partitioning. In some embodiments, all partitions aresubjected to amplification conditions (e.g. reagents and thermalcycling), but amplification only occurs in partitions containing targetnucleic acids (e.g. nucleic acids containing sequences complimentary toprimers added to the sample). In some embodiments, amplification ofnucleic acids in partitioned samples results in some partitionscontaining multiple copies of target nucleic acids and other partitionscontaining no nucleic acids and/or no target nucleic acids (e.g.containing one non-target nucleic acid molecule).

In some embodiments, detection reagents (e.g., fluorescent labels orother optical or non-optical labels) are included with amplificationreagents added to the bulk or partitioned sample. In some embodiments,amplification reagents also serve as detection reagents. In someembodiments, detection reagents are added to partitions followingamplification. In some embodiments, detection reagents comprisefluorescent labels. In some embodiments, amplified target nucleic acids(amplicons) are detectable via detection reagents in their partition. Insome embodiments, unamplified and/or non-target nucleic acid moleculesare not detected. In some embodiments, partitions containing amplifiednucleic acids are detectable using one or more detection reagents (e.g.fluorescent labels). In some embodiments, for example where digitalapproaches are employed, partitions that do not contain amplifiednucleic acid, contain unamplified nucleic acid, and/or contain nonucleic acid are either detectable as such, or are undetectable. In someembodiments, measurements of the relative proportion of target nucleicacids in a sample (e.g. relative to other targets nucleic acids,relative to non-target nucleic acids, relative to total nucleic acids,etc.) or the concentration of target nucleic acids in a sample can bemeasured based on the detection of partitions containing amplifiedtarget sequences.

In some embodiments, following amplification, partitions containingamplified target nucleic acids (amplicons) are sorted from partitionsnot containing amplicons, from partitions not containing nucleic acids,or from amplicons containing other amplified targets. In someembodiments, partitions are sorted based on physical, chemical, and/oroptical characteristics of the partition, the nucleic acids therein(e.g. concentration), and/or labels therein (e.g. fluorescent labels).In some embodiments, individual partitions are isolated for subsequentmanipulation, processing, and/or analysis of the amplicons therein. Insome embodiments, partitions containing similar characteristics (e.g.same fluorescent labels, similar nucleic acid concentrations, etc.) aregrouped (e.g. into packets) for subsequent manipulation, processing,and/or analysis (e.g. of the partitions or of the amplicons therein,etc.).

In some embodiments, amplified and/or sorted nucleic acids arere-amplified to increase amplicon concentration within a partition forsubsequent manipulation, processing, and/or analysis. In someembodiments, amplified and/or sorted nucleic acids are re-amplified toincorporate sequencing reagents into amplicons. In some embodiments,amplified, sorted, and/or re-amplified target nucleic acid molecules aresequenced according to sequencing methods understood in the art. In someembodiment, amplicons are analyzed using compositions and methodsunderstood in the art (e.g. sequencing, mass spectrometry, spectroscopy,hybridization, etc.).

I. Partitioning

In some embodiments, provided herein are systems, devices, and methodsfor dividing volumes of fluid and/or reagents into partitions (e.g.droplets). In some embodiments, the systems, devices, and methodsutilizes partitioning systems, devices, and/or methods. In someembodiments, exemplary partitioning methods and systems include one ormore of emulsification, droplet actuation, microfluidics platforms,continuous-flow microfluidics, reagent immobilization, and combinationsthereof.

In some embodiments, the systems, methods, and devices described hereinfind use for digital PCR, other digital amplification reactions,droplet-based PCR, or other amplification or nucleic acid manipulationreactions employing partitioned, segregated, or transported samples (seee.g., U.S. Pat. Nos., 7,041,481, 7,323,305, 7,842,457, 7,323,305,6,303,343, 7,459,315, 7,888,017, 7,824,889, 7,629,124, 6,586,176,6,664,044, 6,524,456, and 6,309,600, and U.S. Pat. Publ. Nos.20110201526, 20090286687, 20100173293, 20090325184, 20100221713,20080090244, 20100120038, 20110092376, 20110217712, 20110053798,20110244455, 20100248237, 20100092973, 20070195127, 20060153924,20070092914, 20090311713, 20100035323, and 20100227767, hereinincorporated by reference in their entireties). In some embodiments, thesystems, methods, and devices described herein may be integrated into,integrate, or modify existing systems, devices, and methods (see e.g.,U.S. Pat. Nos., 6,337,212, 6,541,274, 7,440,684, 6,524,830, 7,799,553,6,787,338, 7,901,947, 7,439,014, 8,041,463, 7,816,121, 7,851,184,7,709,250, 7,972,778, 7,833,708, 7,285,411, 6,440,722, 6,403,338,6,632,655, 6,881,312, 6,391,559, and 7,622,076, and U.S. Pat. Publ. Nos.20110177563, 20110177587, 20110048951, 20080280331, 20070172954,20110000560, 20080003142, 20100137163, 20100285975, 20080277494, and20100028980, herein incorporated by reference in their entireties).

In some embodiments, partitioning is performed to divide a sample into asufficient number of partitions such that each partition contains one orzero nucleic acid molecules. In some embodiments, partitions areproduced at small enough size such that each partition contains one orzero nucleic acid molecules. In some embodiments, the number and size ofpartitions is based on the concentration and volume of the bulk sample.In some embodiments, the number of nucleic acid molecules to bepartitioned is low, relative to the number of partitions. In someembodiments, based on the relatively low number of target nucleic acidmolecules compared to partitions, the likelihood of a partitioncontaining 2 or more target nucleic acid molecules is low (e.g. 0.1% . .. 0.01% . . . 0.001% . . . 0.0001% . . . 0.00001% . . . 0.000001). Insome embodiments, the number of partitions containing 2 or more nucleicacid molecules is zero. In some embodiments, the number of partitionscontaining 2 or more nucleic acid molecules is essentially zero, or astatistically insignificant fraction of the totally number ofpartitions.

In some embodiments, provided herein are systems, methods, and devicesfor partitioning a bulk volume into partitions (e.g. droplets) byemulsification (Nakano et al. J Biotechnol 2003; 102:117-124.; Margulieset al. Nature 2005; 437:376-380.; herein incorporated by reference intheir entireties). In some embodiments, provided herein are systems andmethods for generating “water-in-oil” droplets (U.S. Pat. App. No.20100173394; herein incorporated by reference in its entirety).

In some embodiments, provided herein are microfluidics systems, methods,and devices for partitioning a bulk volume into partitions (U.S. Pat.App. No. 20100236929; U.S. Pat. App. No. 20100311599; U.S. Pat. App. No.20100163412; U.S. Pat. No. 7,851,184; herein incorporated by referencein their entireties). In some embodiments, microfluidic systems areconfigured to generate monodisperse droplets (Kiss et al. Anal Chem.2008 Dec. 1; 80(23): 8975-8981; herein incorporated by reference in itsentirety). In some embodiments, provided herein are microfluidicssystems for manipulating and/or partitioning samples. In someembodiments, a microfluidics system comprises one or more of channels,valves, pumps, etc. (U.S. Pat. No. 7,842,248, herein incorporated byreference in its entirety). In some embodiments, a microfluidics systemis a continuous-flow microfluidics system (Kopp et al., Science, vol.280, pp. 1046-1048, 1998.; herein incorporated by reference in itsentirety). In some embodiments, microarchitecture includes, but is notlimited to microchannels, microfluidic plates, fixed microchannels,networks of microchannels, internal pumps; external pumps, valves,centrifugal force elements, etc. In some embodiments, themicroarchitecture of the (e.g. droplet microactuator, microfluidicsplatform, and/or continuous-flow microfluidics) is complemented orsupplemented with droplet manipulation techniques, including, but notlimited to electrical (e.g., electrostatic actuation,dielectrophoresis), magnetic, thermal (e.g., thermal Marangoni effects,thermocapillary), mechanical (e.g., surface acoustic waves,micropumping, peristaltic), optical (e.g., opto-electrowetting, opticaltweezers), and chemical means (e.g., chemical gradients). In someembodiments, a droplet microactuator is supplemented with a microfluidisplatform (e.g. continuous flow components) and such combinationapproaches involving discrete droplet operations and microfluidicselements are within the scope of the invention.

In some embodiments, provided herein is a droplet microactuator. In someembodiments, a droplet microactuator is capable of effecting dropletmanipulation and/or operations, such as dispensing, splitting,transporting, merging, mixing, agitating, and the like. In someembodiments the invention employs droplet operation structures andtechniques described in U.S. Pat. No. 6,911,132, entitled “Apparatus forManipulating Droplets by Electrowetting-Based Techniques,” issued onJun. 28, 2005 to Pamula et al.; U.S. patent application Ser. No.11/343,284, entitled “Apparatuses and Methods for Manipulating Dropletson a Printed Circuit Board,” filed on Jan. 30, 2006; U.S. Pat. No.6,773,566, entitled “Electrostatic Actuators for Microfluidics andMethods for Using Same,” issued on Aug. 10, 2004 and U.S. Pat. No.6,565,727, entitled “Actuators for Microfluidics Without Moving Parts,”issued on Jan. 24, 2000, both to Shenderov et al.; U.S. PatentPublication No. 20060254933, entitled “Device for transporting liquidand system for analyzing” published on Nov. 16, 2006 by Adachi et al.,the disclosures of which are incorporated herein by reference in theirentireties. Droplet manipulation is, in some embodiments, accomplishedusing electric field mediated actuation. In such embodiments, electrodesare electronically coupled to a means for controlling electricalconnections to the droplet microactuator. An exemplary dropletmicroactuator includes a substrate including a path and/or array ofelectrodes. In some embodiments, a droplet microactuator includes twoparallel substrates separated by a gap and an array of electrodes on oneor both substrates. One or both of the substrates may be a plate.

II. Amplification

In some embodiments, provided herein are compositions and methods forthe amplification of nucleic acids (e.g. DNA, RNA, etc.). In someembodiments, amplification is performed on a bulk sample of nucleicacids. In some embodiments, amplification is performed on a sample thathas been divided into partitions (e.g. droplets). In some embodiments,an amplification reaction is carried out within each partition. In someembodiments, a partition contains all the reagents necessary for nucleicacid amplification. In some embodiments, amplification is performed on asingle nucleic acid target molecule within a partition. In someembodiments, template nucleic acid is the limiting reagent in apartitioned amplification reaction. In some embodiments, a partitioncontains one or zero target (e.g. template) nucleic acid molecules.

In some embodiments, provided herein are compositions (e.g. primers,buffers, salts, nucleic acid targets, etc.) and methods for theamplification of nucleic acid (e.g. digital droplet amplification, PCRamplification, partitioned amplification, combinations thereof, etc.).In some embodiments, an amplification reaction is any reaction in whichnucleic acid replication occurs repeatedly over time to form multiplecopies of at least one segment of a template or target nucleic acidmolecule (e.g. DNA, RNA). In some embodiments, amplification generatesan exponential or linear increase in the number of copies of thetemplate nucleic acid. Amplifications may produce in excess of a1,000-fold increase in template copy-number and/or target-detectionsignal. Exemplary amplification reactions include, but are not limitedto the polymerase chain reaction (PCR) or ligase chain reaction (LCR),each of which is driven by thermal cycling.

Amplification may be performed with any suitable reagents (e.g. templatenucleic acid (e.g. DNA or RNA), primers, probes, buffers, replicationcatalyzing enzyme (e.g. DNA polymerase, RNA polymerase), nucleotides,salts (e.g. MgCl₂), etc. In some embodiments, an amplification mixtureincludes any combination of at least one primer or primer pair, at leastone probe, at least one replication enzyme (e.g., at least onepolymerase, such as at least one DNA and/or RNA polymerase), anddeoxynucleotide (and/or nucleotide) triphosphates (dNTPs and/or NTPs),etc.

In some embodiments, the systems, devices, and methods utilize nucleicacid amplification that relies on alternating cycles of heating andcooling (i.e., thermal cycling) to achieve successive rounds ofreplication (e.g., PCR). In some embodiments, PCR is used to amplifytarget nucleic acids (e.g. partitioned targets). PCR may be performed bythermal cycling between two or more temperature set points, such as ahigher melting (denaturation) temperature and a lowerannealing/extension temperature, or among three or more temperature setpoints, such as a higher melting temperature, a lower annealingtemperature, and an intermediate extension temperature, among others.PCR may be performed with a thermostable polymerase, such as Taq DNApolymerase (e.g., wild-type enzyme, a Stoffel fragment, FastStartpolymerase, etc.), Pfu DNA polymerase, S-Tbr polymerase, Tth polymerase,Vent polymerase, or a combination thereof, among others. Typical PCRmethods produce an exponential increase in the amount of a productamplicon over successive cycles, although linear PCR methods also finduse.

Any suitable PCR methodology, combination of PCR methodologies, orcombination of amplification techniques may be utilized, such asallele-specific PCR, assembly PCR, asymmetric PCR, digital PCR, endpointPCR, hot-start PCR, in situ PCR, intersequence-specific PCR, inversePCR, linear after exponential PCR, ligation-mediated PCR,methylation-specific PCR, miniprimer PCR, multiplex ligation-dependentprobe amplification, multiplex PCR, nested PCR, overlap-extension PCR,polymerase cycling assembly, qualitative PCR, quantitative PCR,real-time PCR, RT-PCR, single-cell PCR, solid-phase PCR, thermalasymmetric interlaced PCR, touchdown PCR, or universal fast walking PCR,etc.

In some embodiments, provided herein are digital PCR methods. In someembodiments, PCR is performed on portions of a sample (e.g. partitions)to determine the presence or absence, concentration, and/or copy numberof a nucleic acid target in the sample, based on how many of the sampleportions support amplification of the target. In some embodiments, PCRis performed on portions of a sample (e.g. partitions) to detect morethan one target nucleic acid and/or to determine the concentration,and/or relative concentrations of multiple target nucleic acids within asample. In some embodiments, digital PCR is performed as endpoint PCR(e.g., for each of the partitions). In some embodiments, digital PCR isperformed as real-time PCR (e.g., for each of the partitions).

PCR theoretically results in an exponential amplification of a nucleicacid sequence (e.g. template or target nucleic acid) from a sample. Bymeasuring the number of amplification cycles required to achieve athreshold level of amplification (as in real-time PCR), the startingconcentration of nucleic acid can be calculated. However, there are manyfactors the affect the exponential amplification of the PCR process,such as varying amplification efficiencies, presence of PCR inhibitors,low copy numbers of starting nucleic acid, and competition withbackground contaminant nucleic acid. Digital PCR is generallyinsensitive to these factors, since it does not rely on the assumptionthat the PCR process is exponential. In digital PCR, individual nucleicacid molecules are separated from the initial sample into partitions,and then amplified to detectable levels. Each partition then providesdigital information on the presence or absence of each individualnucleic acid molecule within each partition. When enough partitions aremeasured using this technique, the digital information can beconsolidated to make a statistically relevant measure of startingconcentration for the nucleic acid target in the sample. In embodimentsin which multiple target nucleic acids are analyzed, digital PCRprovides statistically relevant measure of the relative concentrationsor ratios to multiple target nucleic acids.

In some embodiments, provided herein is qualitative PCR. In someembodiments, qualitative PCR-based analysis determines whether or not atarget is present in a sample (e.g. whether or not a target is presentin a partition), generally without any substantial quantification oftarget. In some embodiments, digital PCR that is qualitative may beperformed by determining whether a partition or droplet is positive forthe presence of target. In some embodiments, qualitative digital PCR isused to determine the percentage of partitions in a packet that arepositive for the presence of target. In some embodiments, qualitativedigital PCR is used to determine whether a packet of droplets containsat least a threshold percentage of positive droplets (i.e. a positivesample). In some embodiments, qualitative PCR is performed to detect thepresence of multiple targets in a sample.

In some embodiments, the systems, devices, and methods employ RT-PCR(reverse transcription-PCR). In some embodiments, the systems, devices,and methods employ real-time PCR. In some embodiments, the systems,devices, and methods employ endpoint PCR.

III. Amplicon detection

In some embodiments, provided herein are systems, devices, methods, andcompositions to identify the presence of nucleic acids (e.g. amplicons,labeled nucleic acids). In some embodiments, detection involvesmeasurement or detection of a characteristic of an amplified nucleicacid, a component (e.g., droplet or partition) comprising amplifiednucleic acid, or a byproduct of the amplification process, such as aphysical, chemical, luminescence, or electrical aspect, which correlateswith amplification (e.g. fluorescence, pH change, heat change, etc.).

In some embodiments, fluorescence detection methods are provided fordetection of amplified nucleic acid, and/or identification of partitionscontaining amplified nucleic acids. In addition to the reagents alreadydiscussed, and those known to those of skill in the art of nucleic acidamplification and detection, various detection reagents, such asfluorescent and non-fluorescent dyes and probes are provided. Forexample, the protocols may employ reagents suitable for use in a TaqManreaction, such as a TaqMan probe; reagents suitable for use in a SYBRGreen fluorescence detection; reagents suitable for use in a molecularbeacon reaction, such as molecular beacon probes; reagents suitable foruse in a scorpion reaction, such as a scorpion probe; reagents suitablefor use in a fluorescent DNA-binding dye-type reaction, such as afluorescent probe; and/or reagents for use in a LightUp protocol, suchas a LightUp probe. In some embodiments, provided herein are methods andcompositions for detecting and/or quantifying a detectable signal (e.g.fluorescence) from partitions containing amplified target nucleic acid.Thus, for example, methods may employ labeling (e.g. duringamplification, post-amplification) amplified nucleic acids with adetectable label, exposing partitions to a light source at a wavelengthselected to cause the detectable label to fluoresce, and detectingand/or measuring the resulting fluorescence. Fluorescence emitted fromthe partitions can be tracked during amplification reaction to permitmonitoring of the reaction (e.g., using a SYBR Green-type compound), orfluorescence can be measure post-amplification.

In some embodiments, detection of amplified nucleic acids employs one ormore of fluorescent labeling, fluorescent intercalation dyes, FRET-baseddetection methods (U.S. Pat. No. 5,945,283; PCT Publication WO 97/22719;both of which are incorporated by reference in their entireties),quantitative PCR, real-time fluorogenic methods (U.S. Pat. No. 5,210,015to Gelfand, U.S. Pat. No. 5,538,848 to Livak, et al., and U.S. Pat. No.5,863,736 to Haaland, as well as Heid, C. A., et al., Genome Research,6:986-994 (1996); Gibson, U. E. M, et al., Genome Research 6:995-1001(1996); Holland, P. M., et al., Proc. Natl. Acad. Sci. USA 88:7276-7280,(1991); and Livak, K. J., et al., PCR Methods and Applications 357-362(1995), each of which is incorporated by reference in its entirety),molecular beacons (Piatek, A. S., et al., Nat. Biotechnol. 16:359-63(1998); Tyagi, S. and Kramer, F. R., Nature Biotechnology 14:303-308(1996); and Tyagi, S. et al., Nat. Biotechnol. 16:49-53 (1998); hereinincorporated by reference in their entireties), Invader assays (ThirdWave Technologies, (Madison, Wis.)) (Neri, B. P., et al., Advances inNucleic Acid and Protein Analysis 3826:117-125, 2000; hereinincorporated by reference in its entirety), nucleic acid sequence-basedamplification (NASBA; (See, e.g., Compton, J. Nucleic AcidSequence-based Amplification, Nature 350: 91-91, 1991.; hereinincorporated by reference in its entirety), Scorpion probes (Thelwell,et al. Nucleic Acids Research, 28:3752-3761, 2000; herein incorporatedby reference in its entirety), capacitive DNA detection (See, e.g.,Sohn, et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97:10687-10690; hereinincorporated by reference in its entirety), etc.

IV. Amplicon Isolation

In some embodiments, provided herein are methods for sorting and/orisolation of amplified nucleic acid. In some embodiments, providedherein are methods to sort and/or isolate partitions containingamplified nucleic acid. In some embodiments, following amplification oftarget sequences and/or detection of amplicons, partitions containingamplicons are sorted for subsequent manipulation (e.g. re-amplification,labeling, restriction digestion, etc.) and/or analysis (e.g. sequencing,mass detection, etc.).

In some embodiments, amplicons are labeled with detectable and/ormanipulatable labels (e.g. fluorescent dyes), during or afteramplification, by accepted methods understood to those in the art (e.g.,intercalation, incorporation, hybridization, etc.) In some embodiments,partitions containing labeled amplicons are detected and/or sorted (e.g.segregated from non-amplicon-containing partitions, grouped according topresence of a particular label, etc.). For example, in some embodiments,amplicon-containing partitions are mechanically separated bymicro-manipulators, electrophoresis, flow cytometry, or other sortingtechniques known to those in the art. The following references provideguidance for selecting means for analyzing and/or sortingmicroparticles: Pace, U.S. Pat. No. 4,908,112; Saur et al., U.S. Pat.No. 4,710,472; Senyei et al., U.S. Pat. No. 4,230,685; Wilding et al.,U.S. Pat. No. 5,637,469; Penniman et al., U.S. Pat. No. 4,661,225;Kamaukhov et al., U.S. Pat. No. 4,354,114; Abbott et al., U.S. Pat. No.5,104,791; Gavin et al., PCT publication WO 97/40383; hereinincorporated by reference in their entireties.

In some embodiments, partitions containing fluorescently labeled DNAstrands are detected, classified, isolated, and/or sorted byfluorescence-activated cell sorting (FACS; See, e.g., Van Dilla et al.,Flow Cytometry: Instrumentation and Data Analysis (Academic Press, NewYork, 1985); Fulwyler et al., U.S. Pat. No. 3,710,933; Gray et al., U.S.Pat. No. 4,361,400; Dolbeare et al., U.S. Pat. No. 4,812,394; hereinincorporated by reference in their entireties. In some embodiments,amplicons are fluorescently labeled with detectable and/or manipulatablefluorescent labels, during or after amplification, by accepted methodsunderstood to those in the art (e.g., intercalation, incorporation,hybridization, etc.). In some embodiments, upon excitation with one ormore high intensity light sources, such as a laser, a mercury arc lamp,or the like, each partition containing amplified (and labeled) targetnucleic acids will generate fluorescent signals. In some embodiments,partitions exhibiting fluorescence above background, or above athreshold level, are sorted by a FACS instrument, according to methodsunderstood by those of skill in the art. Thus, in some embodiments,partitions are sorted according to their relative optical signal, andcollected for further analysis by accumulating those partitionsgenerating a signal within a predetermined range of values correspondingto the presence of amplified target nucleic acid. In some embodiments,partitions are sorted and transferred to reaction vessels and/orplatforms suitable for subsequent manipulation, processing, and/oranalysis.

V. Analysis

Amplified nucleic acid molecules may be analyzed by any number oftechniques to determine the presence of, amount of, or identity of themolecule. Non-limiting examples include sequencing, mass determination,and base composition determination. The analysis may identify thesequence of all or a part of the amplified nucleic acid or one or moreof its properties or characteristics to reveal the desired information.For example, in some embodiments, the presence of a polymorphism isdetermined, for example, to provide information about the nature of anorganism (e.g., pathogen), a disease state, a metabolic state, and thelike. In some embodiments, the methylation status of a nucleic acid isdetermined. In some such embodiments, a target nucleic acid may bechemically modified (e.g., via bisulfate treatment) prior toamplification to create a marker for the methylation sites.

Illustrative non-limiting examples of nucleic acid sequencing techniquesinclude, but are not limited to, chain terminator (Sanger) sequencingand dye terminator sequencing, as well as “next generation” sequencingtechniques. Those of ordinary skill in the art will recognize thatbecause RNA is less stable in the cell and more prone to nuclease attackexperimentally RNA is usually, although not necessarily, reversetranscribed to DNA before sequencing.

A number of DNA sequencing techniques are known in the art, includingfluorescence-based sequencing methodologies (See, e.g., Birren et al.,Genome Analysis: Analyzing DNA, 1, Cold Spring Harbor, N.Y.; hereinincorporated by reference in its entirety). In some embodiments,automated sequencing techniques understood in that art are utilized. Insome embodiments, the systems, devices, and methods employ parallelsequencing of partitioned amplicons (PCT Publication No: WO2006084132 toKevin McKernan et al., herein incorporated by reference in itsentirety). In some embodiments, DNA sequencing is achieved by paralleloligonucleotide extension (See, e.g., U.S. Pat. No. 5,750,341 toMacevicz et al., and U.S. Pat. No. 6,306,597 to Macevicz et al., both ofwhich are herein incorporated by reference in their entireties).Additional examples of sequencing techniques include the Church polonytechnology (Mitra et al., 2003, Analytical Biochemistry 320, 55-65;Shendure et al., 2005 Science 309, 1728-1732; U.S. Pat. No. 6,432,360,U.S. Pat. No. 6,485,944, U.S. Pat. No. 6,511,803; herein incorporated byreference in their entireties) the 454 picotiter pyrosequencingtechnology (Margulies et al., 2005 Nature 437, 376-380; US 20050130173;herein incorporated by reference in their entireties), the Solexa singlebase addition technology (Bennett et al., 2005, Pharmacogenomics, 6,373-382; U.S. Pat. No. 6,787,308; U.S. Pat. No. 6,833,246; hereinincorporated by reference in their entireties), the Lynx massivelyparallel signature sequencing technology (Brenner et al. (2000). Nat.Biotechnol. 18:630-634; U.S. Pat. No. 5,695,934; U.S. Pat. No.5,714,330; herein incorporated by reference in their entireties) and theAdessi PCR colony technology (Adessi et al. (2000). Nucleic Acid Res.28, E87; WO 00018957; herein incorporated by reference in its entirety).

In some embodiments, chain terminator sequencing is utilized. Chainterminator sequencing uses sequence-specific termination of a DNAsynthesis reaction using modified nucleotide substrates. Extension isinitiated at a specific site on the template DNA by using a shortradioactive, or other labeled, oligonucleotide primer complementary tothe template at that region. The oligonucleotide primer is extendedusing a DNA polymerase, standard four deoxynucleotide bases, and a lowconcentration of one chain terminating nucleotide, most commonly adi-deoxynucleotide. This reaction is repeated in four separate tubeswith each of the bases taking turns as the di-deoxynucleotide. Limitedincorporation of the chain terminating nucleotide by the DNA polymeraseresults in a series of related DNA fragments that are terminated only atpositions where that particular di-deoxynucleotide is used. For eachreaction tube, the fragments are size-separated by electrophoresis in aslab polyacrylamide gel or a capillary tube filled with a viscouspolymer. The sequence is determined by reading which lane produces avisualized mark from the labeled primer as you scan from the top of thegel to the bottom.

Dye terminator sequencing alternatively labels the terminators. Completesequencing can be performed in a single reaction by labeling each of thedi-deoxynucleotide chain-terminators with a separate fluorescent dye,which fluoresces at a different wavelength.

A set of methods referred to as “next-generation sequencing” techniqueshave emerged as alternatives to Sanger and dye-terminator sequencingmethods (Voelkerding et al., Clinical Chem., 55: 641-658, 2009; MacLeanet al., Nature Rev. Microbiol., 7: 287-296; each herein incorporated byreference in their entirety). Next-generation sequencing (NGS) methodsshare the common feature of massively parallel, high-throughputstrategies, with the goal of lower costs in comparison to oldersequencing methods. NGS methods can be broadly divided into those thatrequire template amplification and those that do not.Amplification-requiring methods include pyrosequencing commercialized byRoche as the 454 technology platforms (e.g., GS 20 and GS FLX), theSolexa platform commercialized by Illumina, and the SupportedOligonucleotide Ligation and Detection (SOLiD) platform commercializedby Applied Biosystems. Non-amplification approaches, also known assingle-molecule sequencing, are exemplified by the HeliScope platformcommercialized by Helicos BioSciences, and emerging platformscommercialized by VisiGen, Oxford Nanopore Technologies Ltd., andPacific Biosciences, respectively.

In pyrosequencing (Voelkerding et al., Clinical Chem., 55: 641-658,2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296; U.S. Pat. No.6,210,891; U.S. Pat. No. 6,258,568; each herein incorporated byreference in its entirety), template DNA is fragmented, end-repaired,ligated to adaptors, and clonally amplified in-situ by capturing singletemplate molecules with beads bearing oligonucleotides complementary tothe adaptors. Each bead bearing a single template type iscompartmentalized into a water-in-oil microvesicle, and the template isclonally amplified using a technique referred to as emulsion PCR. Theemulsion is disrupted after amplification and beads are deposited intoindividual wells of a picotiter plate functioning as a flow cell duringthe sequencing reactions. Ordered, iterative introduction of each of thefour dNTP reagents occurs in the flow cell in the presence of sequencingenzymes and luminescent reporter such as luciferase. In the event thatan appropriate dNTP is added to the 3′ end of the sequencing primer, theresulting production of ATP causes a burst of luminescence within thewell, which is recorded using a CCD camera. It is possible to achieveread lengths greater than or equal to 400 bases, and 1×10⁶ sequencereads can be achieved, resulting in up to 500 million base pairs (Mb) ofsequence.

In the Solexa/Illumina platform (Voelkerding et al., Clinical Chem., 55:641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296; U.S.Pat. No. 6,833,246; U.S. Pat. No. 7,115,400; U.S. Pat. No. 6,969,488;each herein incorporated by reference in its entirety), sequencing dataare produced in the form of shorter-length reads. In this method,single-stranded fragmented DNA is end-repaired to generate5′-phosphorylated blunt ends, followed by Klenow-mediated addition of asingle A base to the 3′ end of the fragments. A-addition facilitatesaddition of T-overhang adaptor oligonucleotides, which are subsequentlyused to capture the template-adaptor molecules on the surface of a flowcell that is studded with oligonucleotide anchors. The anchor is used asa PCR primer, but because of the length of the template and itsproximity to other nearby anchor oligonucleotides, extension by PCRresults in the “arching over” of the molecule to hybridize with anadjacent anchor oligonucleotide to form a bridge structure on thesurface of the flow cell. These loops of DNA are denatured and cleaved.Forward strands are then sequenced with reversible dye terminators. Thesequence of incorporated nucleotides is determined by detection ofpost-incorporation fluorescence, with each fluor and block removed priorto the next cycle of dNTP addition. Sequence read length ranges from 36nucleotides to over 50 nucleotides, with overall output exceeding 1billion nucleotide pairs per analytical run.

Sequencing nucleic acid molecules using SOLiD technology (Voelkerding etal., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev.Microbiol., 7: 287-296; U.S. Pat. No. 5,912,148; U.S. Pat. No.6,130,073; each herein incorporated by reference in their entirety) alsoinvolves fragmentation of the template, ligation to oligonucleotideadaptors, attachment to beads, and clonal amplification by emulsion PCR.Following this, beads bearing template are immobilized on a derivatizedsurface of a glass flow-cell, and a primer complementary to the adaptoroligonucleotide is annealed. However, rather than utilizing this primerfor 3′ extension, it is instead used to provide a 5′ phosphate group forligation to interrogation probes containing two probe-specific basesfollowed by 6 degenerate bases and one of four fluorescent labels. Inthe SOLiD system, interrogation probes have 16 possible combinations ofthe two bases at the 3′ end of each probe, and one of four fluors at the5′ end. Fluor color and thus identity of each probe corresponds tospecified color-space coding schemes. Multiple rounds (usually 7) ofprobe annealing, ligation, and fluor detection are followed bydenaturation, and then a second round of sequencing using a primer thatis offset by one base relative to the initial primer. In this manner,the template sequence can be computationally re-constructed, andtemplate bases are interrogated twice, resulting in increased accuracy.Sequence read length averages 35 nucleotides, and overall output exceeds4 billion bases per sequencing run.

In certain embodiments, nanopore sequencing in employed (see, e.g.,Astier et al., J Am Chem Soc. 2006 Feb. 8; 128(5):1705-10, hereinincorporated by reference). The theory behind nanopore sequencing has todo with what occurs when the nanopore is immersed in a conducting fluidand a potential (voltage) is applied across it: under these conditions aslight electric current due to conduction of ions through the nanoporecan be observed, and the amount of current is exceedingly sensitive tothe size of the nanopore. If DNA molecules pass (or part of the DNAmolecule passes) through the nanopore, this can create a change in themagnitude of the current through the nanopore, thereby allowing thesequences of the DNA molecule to be determined.

Another exemplary nucleic acid sequencing approach that may be adaptedfor use with the systems, devices, and methods was developed by StratosGenomics, Inc. and involves the use of Xpandomers. This sequencingprocess typically includes providing a daughter strand produced by atemplate-directed synthesis. The daughter strand generally includes aplurality of subunits coupled in a sequence corresponding to acontiguous nucleotide sequence of all or a portion of a target nucleicacid in which the individual subunits comprise a tether, at least oneprobe or nucleobase residue, and at least one selectively cleavablebond. The selectively cleavable bond(s) is/are cleaved to yield anXpandomer of a length longer than the plurality of the subunits of thedaughter strand. The Xpandomer typically includes the tethers andreporter elements for parsing genetic information in a sequencecorresponding to the contiguous nucleotide sequence of all or a portionof the target nucleic acid. Reporter elements of the Xpandomer are thendetected. Additional details relating to Xpandomer-based approaches aredescribed in, for example, U.S. Patent Publication No. 20090035777,entitled “HIGH THROUGHPUT NUCLEIC ACID SEQUENCING BY EXPANSION,” thatwas filed Jun. 19, 2008, which is incorporated herein in its entirety.

Other emerging single molecule sequencing methods include real-timesequencing by synthesis using a VisiGen platform (Voelkerding et al.,Clinical Chem., 55: 641- 658, 2009 ; U.S. Pat. No. 7,329,492; U.S.patent application Ser. No. 11/671956; U.S. patent application Ser. No.11/781166; each herein incorporated by reference in their entirety) inwhich immobilized, primed DNA template is subjected to strand extensionusing a fluorescently-modified polymerase and florescent acceptormolecules, resulting in detectible fluorescence resonance energytransfer (FRET) upon nucleotide addition.

Processes and systems for such real time sequencing that may be adaptedfor use with the invention are described in, for example, U.S. Pat. No.7,405,281, entitled “Fluorescent nucleotide analogs and uses therefor”,issued Jul. 29, 2008 to Xu et al., U.S. Pat. No. 7,315,019, entitled“Arrays of optical confinements and uses thereof”, issued Jan. 1, 2008to Turner et al., U.S. Pat. No. 7,313,308, entitled “Optical analysis ofmolecules”, issued Dec. 25, 2007 to Turner et al., U.S. Pat. No.7,302,146, entitled “Apparatus and method for analysis of molecules” ,issued Nov. 27, 2007 to Turner et al., and U.S. Pat. No. 7,170,050,entitled “Apparatus and methods for optical analysis of molecules” ,issued Jan. 30, 2007 to Turner et al., U.S. Patent Publications Nos.20080212960, entitled “Methods and systems for simultaneous real-timemonitoring of optical signals from multiple sources”, filed Oct. 26,2007 by Lundquist et al., 20080206764, entitled “Flowcell system forsingle molecule detection”, filed Oct. 26, 2007 by Williams et al.,20080199932, entitled “Active surface coupled polymerases”, filed Oct.26, 2007 by Hanzel et al., 20080199874, entitled “CONTROLLABLE STRANDSCISSION OF MINI CIRCLE DNA”, filed Feb. 11, 2008 by Otto et al.,20080176769, entitled “Articles having localized molecules disposedthereon and methods of producing same”, filed Oct. 26, 2007 by Rank etal., 20080176316, entitled “Mitigation of photodamage in analyticalreactions”, filed Oct. 31, 2007 by Eid et al., 20080176241, entitled“Mitigation of photodamage in analytical reactions”, filed Oct. 31, 2007by Eid et al., 20080165346, entitled “Methods and systems forsimultaneous real-time monitoring of optical signals from multiplesources”, filed Oct. 26, 2007 by Lundquist et al., 20080160531, entitled“Uniform surfaces for hybrid material substrates and methods for makingand using same”, filed Oct. 31, 2007 by Korlach, 20080157005, entitled“Methods and systems for simultaneous real-time monitoring of opticalsignals from multiple sources”, filed Oct. 26, 2007 by Lundquist et al.,20080153100, entitled “Articles having localized molecules disposedthereon and methods of producing same”, filed Oct. 31, 2007 by Rank etal., 20080153095, entitled “CHARGE SWITCH NUCLEOTIDES”, filed Oct. 26,2007 by Williams et al., 20080152281, entitled “Substrates, systems andmethods for analyzing materials”, filed Oct. 31, 2007 by Lundquist etal., 20080152280, entitled “Substrates, systems and methods foranalyzing materials”, filed Oct. 31, 2007 by Lundquist et al.,20080145278, entitled “Uniform surfaces for hybrid material substratesand methods for making and using same”, filed Oct. 31, 2007 by Korlach,20080128627, entitled “SUBSTRATES, SYSTEMS AND METHODS FOR ANALYZINGMATERIALS”, filed Aug. 31, 2007 by Lundquist et al., 20080108082,entitled “Polymerase enzymes and reagents for enhanced nucleic acidsequencing”, filed Oct. 22, 2007 by Rank et al., 20080095488, entitled“SUBSTRATES FOR PERFORMING ANALYTICAL REACTIONS”, filed Jun. 11, 2007 byFoquet et al., 20080080059, entitled “MODULAR OPTICAL COMPONENTS ANDSYSTEMS INCORPORATING SAME”, filed Sep. 27, 2007 by Dixon et al.,20080050747, entitled “Articles having localized molecules disposedthereon and methods of producing and using same”, filed Aug. 14, 2007 byKorlach et al., 20080032301, entitled “Articles having localizedmolecules disposed thereon and methods of producing same”, filed Mar.29, 2007 by Rank et al., 20080030628, entitled “Methods and systems forsimultaneous real-time monitoring of optical signals from multiplesources”, filed Feb. 9, 2007 by Lundquist et al., 20080009007, entitled“CONTROLLED INITIATION OF PRIMER EXTENSION”, filed Jun. 15, 2007 by Lyleet al., 20070238679, entitled “Articles having localized moleculesdisposed thereon and methods of producing same”, filed Mar. 30, 2006 byRank et al., 20070231804, entitled “Methods, systems and compositionsfor monitoring enzyme activity and applications thereof”, filed Mar. 31,2006 by Korlach et al., 20070206187, entitled “Methods and systems forsimultaneous real-time monitoring of optical signals from multiplesources”, filed Feb. 9, 2007 by Lundquist et al., 20070196846, entitled“Polymerases for nucleotide analogue incorporation”, filed Dec. 21, 2006by Hanzel et al., 20070188750, entitled “Methods and systems forsimultaneous real-time monitoring of optical signals from multiplesources”, filed Jul. 7, 2006 by Lundquist et al., 20070161017, entitled“MITIGATION OF PHOTODAMAGE IN ANALYTICAL REACTIONS”, filed Dec. 1, 2006by Eid et al., 20070141598, entitled “Nucleotide Compositions and UsesThereof”, filed Nov. 3, 2006 by Turner et al., 20070134128, entitled“Uniform surfaces for hybrid material substrate and methods for makingand using same”, filed Nov. 27, 2006 by Korlach, 20070128133, entitled“Mitigation of photodamage in analytical reactions”, filed Dec. 2, 2005by Eid et al., 20070077564, entitled “Reactive surfaces, substrates andmethods of producing same”, filed Sep. 30, 2005 by Roitman et al.,20070072196, entitled “Fluorescent nucleotide analogs and usestherefore”, filed Sep. 29, 2005 by Xu et al., and 20070036511, entitled“Methods and systems for monitoring multiple optical signals from asingle source”, filed Aug. 11, 2005 by Lundquist et al., and Korlach etal. (2008) “Selective aluminum passivation for targeted immobilizationof single DNA polymerase molecules in zero-mode waveguidenanostructures” Proc. Natl. Acad. Sci. U.S.A. 105(4): 11761181—all ofwhich are herein incorporated by reference in their entireties.

In some embodiments, nucleic acids are analyzed by determination oftheir mass and/or base composition. For example, in some embodiments,nucleic acids are detected and characterized by the identification of aunique base composition signature (BCS) using mass spectrometry (e.g.,Abbott PLEX-ID system, Abbot Ibis Biosciences, Abbott Park, Ill.)described in U.S. Pat. Nos. 7,108,974, 8,017,743, and 8,017,322; each ofwhich is herein incorporated by reference in its entirety.

In some embodiments, a MassARRAY system (Sequenom, San Diego, Calif.) isused to detect or analyze sequences (See e.g., U.S. Pat. Nos. 6,043,031;5,777,324; and 5,605,798; each of which is herein incorporated byreference).

VI. Samples

The amplification methods, compositions, systems, and devices make useof samples which include a nucleic acid template. Samples may be derivedfrom any suitable source, and for purposes related to any field,including but not limited to diagnostics, research, forensics,epidemiology, pathology, archaeology, etc. A sample may be biological,environmental, forensic, veterinary, clinical, etc. in origin. Samplesmay include nucleic acid derived from any suitable source, includingeukaryotes, prokaryotes (e.g. infectious bacteria), mammals, humans,non-human primates, canines, felines, bovines, equines, porcines, mice,viruses, etc. Samples may contain, e.g., whole organisms, organs,tissues, cells, organelles (e.g., chloroplasts, mitochondria), syntheticnucleic acid, cell lysate, etc. Nucleic acid present in a sample (e.g.target nucleic acid, template nucleic acid, non-target nucleic acid,contaminant nucleic acid may be of any type, e.g., genomic DNA, RNA,microRNA, mitochondrial DNA, plasmids, bacteriophages, viruses,synthetic origin, natural origin, and/or artificial sequences(non-naturally occurring), synthetically-produced but naturallyoccurring sequences, etc. Biological specimens may, for example, includewhole blood, lymphatic fluid, serum, plasma, sweat, tear, saliva,sputum, cerebrospinal (CSF) fluids, amniotic fluid, seminal fluid,vaginal excretions, serous fluid, synovial fluid, pericardial fluid,peritoneal fluid, pleural fluid, transudates, exudates, cystic fluid,bile, urine, gastric fluids, intestinal fluids, fecal samples, aspirates(bone marrow, fine needle) and swabs or washes (e.g., oral,nasopharangeal, bronchial, bronchioalveolar, optic, rectal, intestinal,vaginal, epidermal, etc.) and/or other fresh, frozen, cultured,preserved (PAXgene™, RNAlater™, RNasin®, etc.) or archived biologicalspecimens (formalin fixed paraffin-embedded (FFPE), fixedcell/lymphocyte pellet, etc.).

In some embodiments, samples are mixed samples (e.g. containing mixednucleic acid populations). In some embodiments, samples analyzed bymethods herein contain, or may contain, a plurality of different nucleicacid sequences. In some embodiments, a sample (e.g. mixed sample)contains one or more nucleic acid molecules (e.g. 1 . . . 10 . . . 10² .. . 10³ . . . 10⁴ . . . 10⁵ . . . 10⁶ . . . 10⁷, etc.) that contain atarget sequence of interest in a particular application. In someembodiments, a sample (e.g. mixed sample) contains zero nucleic acidmolecules that contain a target sequence of interest in a particularapplication. In some embodiments, a sample (e.g. mixed sample) containsnucleic acid molecules with a plurality of different sequences that allcontain a target sequence of interest. In some embodiments, a sample(e.g. mixed sample) contains one or more nucleic acid molecules (e.g. 1. . . 10 . . . 10² . . . 10³ . . . 10⁴ . . . 10⁵ . . . 10⁶ . . . 10⁷ ,etc.) that do not contain a target sequence of interest in a particularapplication. In some embodiments, a sample (e.g. mixed sample) containszero nucleic acid molecules that do not contain a target sequence ofinterest in a particular application. In some embodiments, a sample(e.g. mixed sample) contains nucleic acid molecules with a plurality ofdifferent sequences that do not contain a target sequence of interest.In some embodiments, a sample contains more nucleic acid molecules thatdo not contain a target sequence than nucleic acid molecules that docontain a target sequence (e.g. 1.01:1 . . . 2:1 . . . 5:1 . . . 10:1 .. . 20:1 . . . 50:1 . . . 10^(2:)1 . . . 10³:1 . . . 10⁴:1 . . . 10⁵:1 .. . 10⁶:1 . . . 10⁷:1). In some embodiments, a sample contains morenucleic acid molecules that do contain a target sequence than nucleicacid molecules that do not contain a target sequence (e.g. 1.01:1 . . .2:1 . . . 5:1 . . . 10:1 . . . 20:1 . . . 50:1 . . . 10²:1 . . . 10³:1 .. . 10⁴:1 . . . 10⁵:1 . . . 10⁶:1 . . . 10⁷:1). In some embodiments, asample contains a single target sequence that may be present in one ormore nucleic acid molecules in the sample. In some embodiments, a samplecontains two or more target sequences (e.g. 2, 3, 4, 5 . . . 10 . . . 20. . . 50 . . . 100, etc.)that may each be present in one or more nucleicacid molecules in the sample.

In some embodiments, various sample processing steps may be accomplishedto prepare the nucleic acid molecules within a sample, including, butnot limited to cell lysis, restriction digestion, purification,precipitation, resuspension (e.g. in amplification buffer), dialysis,etc. In some embodiments, sample processing is performed before or afterany of the steps including, but not limited to partitioning,amplification, re-amplification), amplicon detection, ampliconisolation, sequencing, etc.

All publications and patents mentioned in the present application areherein incorporated by reference. Various modification and variation ofthe described methods and compositions of the invention will be apparentto those skilled in the art without departing from the scope and spiritof the invention. Although the invention has been described inconnection with specific preferred embodiments, it should be understoodthat the invention as claimed should not be unduly limited to suchspecific embodiments. Indeed, various modifications of the describedmodes for carrying out the invention that are obvious to those skilledin the relevant fields are intended to be within the scope of thefollowing claim.

We claim:
 1. A method comprising: a) providing a device comprising: i) amicrochannel comprising a surface configured for transmission a carriermedium comprising droplets, ii) a first thermal layer positioned alongsaid microchannel configured to heat a portion of said microchannelsurface to a nucleic acid denaturation temperature; and iii) a secondthermal layer positioned along said microchannel configured to heat orcool a portion of said microchannel surface to a nucleic acid annealingtemperature; wherein said microchannel and first and second thermallayers are positioned to create a thermal gradient across across-section of said microchannel, and wherein said thermal gradientcomprises a nucleic acid denaturation zone, a nucleic acid annealingzone, and a nucleic acid polymerization zone; and b) flowing saidcarrier medium through said microchannel such that said dropletsrepeatedly pass through said thermal gradient causing temperaturecycling in said droplets, wherein said droplets each comprise primers, anucleic acid template, a polymerase, and nucleotides, and wherein saidtemperature cycling causes amplification of said nucleic acid templatesin said droplets.
 2. The method of claim 1, further comprisingsequencing the amplified templates in at least some of the droplets. 3.The method of claim 1, wherein flowing said carrier medium through saidmicrochannel is performed in a continuous manner, a pulsed manner, abatch manner, or fed-batch manner.